title: [Identification of hepatitis B virus YMDD point mutation using peptide nucleic acid clamping PCR].
abstract: OBJECTIVE
  To establish a peptide nucleic acid clamping PCR assay for detecting hepatitis B virus (HBV) drug resistance mutation.
  
  
  METHODS
  RtM204I (ATT) mutant, rtM204V (GTG) mutant and rtM204 (ATG) wild-type plasmids mixed at different ratios were detected for mutations by PNA clamping PCR assay and direct sequencing, and the sensitivity and specificity of the two methods were compared. Serum samples from 85 patients with chronic HBV infection were detected for drug resistance using the two methods.
  
  
  RESULTS
  The sensitivity of PNA-PCR assay was 0.001% in a 10(5)-fold excess of wild-type HBV DNA with a detection limit of 10(1) copies. The sensitivity of direct sequencing was 10% with a detection limit of 10(4) copies. Mutants were detected in 73 of the 85 serum samples (85.9%), including YIDD in 40 samples, YVDD in 23 samples, and YIDD+YVDD in 10 samples. The agreement of PNA-PCR assay with direct sequencing was only 40% (34/85, YIDD in 21 samples, YVDD in 11 samples, and YIDD+YVDD in 2 samples). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity.
  
  
  CONCLUSIONS
  PNA-PCR assay appears to be a more sensitive and rapid assay for detection of HBV genotypic resistance.
authors.affiliation: Depatment of Infectous Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. tyjs05yingying@163.com
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Haitang
authors.initials: H
authors.lastname: He
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Jinlin
authors.initials: J
authors.lastname: Hou
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral